Iron-sulfur (FeS) clusters are ancient ubiquitous metalloprotein cofactors whose origins are thought to lay in the reducing environment of the early anaerobic biosphere. The transition to aerobic life has created several problems for FeS clusters by both limiting the bio-availability of iron due to oxidation and directly promoting the destruction of clusters with oxygen species such as superoxide and peroxide, which are by-products of aerobic respiration. This susceptibility has been leveraged by host defense mechanisms, which target FeS clusters as components through which to promote cell death. The components of FeS clusters, iron and sulfur, are highly toxic to cells and FeS clusters are formed by specific biosynthesis pathways such as the iscRSUhscBAfdx operon and sufABCDSE operon in Escherichia coli. Our long term goal is to determine a complete set of factors involved in FeS cluster biosynthesis in the model prokaryote E. coli and to functionally characterize their roles in FeS cluster formation. The objective of the proposed research is the characterization of a novel factor, the CGFS-type monothiol glutaredoxin, GrxD, which has been implicated as functioning with the E. coli Suf FeS biosynthesis pathway, and the determination of its role in E. coli. The Suf pathway appears to have adapted to a role in FeS cluster synthesis under stress conditions, such as oxidative stress and iron limitation, and has been shown to be necessary for complete virulence of the plant pathogen Erwinia chrysanthemi. Moreover, the process of FeS cluster biosynthesis is an essential trait, and the Suf system has been identified as the only FeS cluster biosynthesis pathway in some bacterial species, such as the pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis kills approximately 2 million people a year, mainly in the developing world. The Suf system has been characterized in some detail in recent years, however, studies of both Isc and Suf systems have yet to identify specific adaptations which make Suf capable of FeS cluster synthesis under stress conditions. Moreover, recent identification of factors outside of these two core operons, which participate in FeS cluster biosynthesis, suggests that other factors not encoded in the Suf operon, such as GrxD, may work with the Suf system and facilitate the synthesis of FeS clusters under stress conditions. Using a combination of biochemical assays, functional genomics, protein chemistry and molecular biology our aims are to (i) determine the basis of synthetic lethality between grxD and Isc system mutants, and if these genetic interactions can be alleviated under specific conditions, (ii) characterize the physical interactions of GrxD and determine the composition of protein complexes in which it participates, and the effect of binding partners on the biochemical properties of GrxD, (iii) specifically assess the ability of GrxD to transfer its FeS cluster, and (iv) determine the functional association of GrxD to both Suf and Isc systems.